1. Field of the Invention
The present invention generally relates to amino acid sequences that can interact with specified glutamate receptors. In one aspect, the invention features purified polypeptides that can interact with the glutamate receptors. In another aspect, the invention relates to isolated polynucleotides that encode the polypeptides. The present invention has a variety of applications, including use in detecting disorders in the nervous or reproductive system of a subject.
2. Background
The synaptic cytoskeleton plays a critical role in the formation and maintenance of synapses in the nervous system. Recent studies have identified a new protein motif called a PDZ domain which may be important in the targeting of proteins to cell-cell junctions. PDZ domains within cytoskeleton associated proteins mediate the interaction of the cytoskeleton with the C-termini of a variety of membrane proteins.
Synapses are specialized areas of cell-cell contact that are optimized for the efficient transduction of signals between neurons in the brain. Both the pre- and postsynaptic membrane have an elaborate cytoarchitecture that is essential for the functional organization of the synapse. Neurons have many different types of synapses and the synaptic cytoskeleton is likely to play an important role in the localization of different receptors and ion channels to their appropriate synapses. A variety of studies on the neuromuscular junction and inhibitory synapses in spinal cord have shown that the cytoskeleton is intimately involved in the clustering of synaptic components at the postsynaptic membrane [Froehner, Annul Rev. Neurosci., 16:347-368 (1993), Hall et al., Cell/Neuron, 72:99-121 (1993), Gautam et al., Nature, 377:232-236 (1995), Kirsch et al., Nature, 266:745-748 (1993), Meyer et al., Neuron, 15:563-572 (1993), Kirsch et al., J. Neuroscience, 15:41484156 (1995)] For example, the synaptic peripheral membrane proteins rapsyn and gephryin have been shown to play a critical role in the synaptic targeting of nicotinic acetylcholine receptors and glycine receptors, respectively [Froehner, Annul Rev. Neurosci., 16:347-368 (1993), Hall et al., Cell/Neuron, 72:99-121 (1993), Gautam et al., Nature, 377:232-236 (1995), Kirsch et al., Nature, 266:745-748 (1993), Meyer et al., Neuron, 15:563-572 (1995), Kirsch et al., J. Neuroscience, 15:41484156 (1995)]. The cytoskeletal proteins involved in the formation of excitatory synapses in the central nervous system, however, have only recently begun to be identified [Kornau et al., Science, 269: 1737-1740 (1995), Niethammer et al., J. Neurosci, 16:2157-2163 (1996), Ehlers et al., Curr. Opin. in Cell Biol., 8:490495 (1996)].
Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission in the central nervous system and play important roles in the synaptic plasticity underlying learning and memory and neural development, and in the excitotoxicity associated with stroke and other neurological disorders [Hollmann et al., Annul Rev. Neurosci., 17:31-108 (1994), Seeburg, The TINS/TIPS Lecture. Trends Neurosci., 16:359-365 (1993), Bliss et al., Nature, 361:31-39 (1993), Linden, Neuron, 12:457472 (1994), Choi, Trends Neurosci., 18:58-60 (1995)]. These receptors can be divided into three different subclasses, AMPA (.alpha.-amino-3-hydroxy-5-methyl-isoxazole4-propionic acid), kainate and NMDA (N-methyl-D-aspartate) receptors, based on their physiological and pharmacological properties [Hollmann et al., Annul Rev. Neurosci., 17:31-108 (1994), Seeburg, The TINS/TIPS Lecture. Trends Neurosci., 16:359-365 (1993)]. AMPA and kainate receptors mediate rapid synaptic transmission while NMDA receptors are important in activity-dependent plasticity in the nervous system and in excitotoxicity. These receptors are ligand-gated ion channels consisting of oligomeric complexes of homologous subunits [Hollmann et al., Annul Rev. Neurosci., 17:31-108 (1994), Seeburg., The TINS/TIPS Lecture. Trends Neurosci., 16:359-365 (1993)]. Molecular cloning studies have demonstrated that each receptor subclass is composed of several distinct subunits. The GluR14 subunits belong to the AMPA subclass, the GluR5-7 and KA1-2 subunits belong to the kainate subclass, while the NR1 and NR2A-D subunits belong to the NMDA subclass of ionotropic glutamate receptors [Hollmann et al., Annul Rev. Neurosci., 17:31-108 (1994), Seeburg, The TINS/TIPS Lecture. Trends Neurosci., 16:359-365 (1993)]. These subunits have been proposed to have a large extracellular N-terminal domain, three transmembrane domains, and an intracellular C-terminal domain [Molnar et al., Neuroscience, 53:307-326 (1993), Tingley et al., Nature, 364:70-73 (1993), Wo et al., Proc. Natl. Acad. Sci. USA, 91:7154-7158 (1994), Hollmann et al., Neuron, 13:1331-1343 (1994), Stem-Bach et al., Neuron, 13:1345-1357 (1994), Bennett et al., Neuron, 14:373-384 (1995), Wood et al., Proc. Natl. Acad. Sci., USA, 92:48824886 (1995), Roche et al., Neuron, 16:1179-1188 (1996)] Glutamate receptor diversity is generated through the differential combination of subunits as well as alternative splicing and editing of glutamate receptor mRNAs [Hollmann et al., Annul Rev. Neurosci., 17:31-108 (1994), Seeburg, The TINS/TIPS Lecture. Trends Neurosci., 16:359-365 (1993)].
Iontophoretic mapping, immunocytochemistry and immunoelectron microscopy studies have demonstrated that ionotropic glutamate receptors are specifically localized to the postsynaptic membranes of CNS neurons [Jones et al., Neuron, 7:593-603 (1991), Petralia et al., Neurol. 318:329-354 (1992), Craig et al., Neuron, 10:1055-1068 (1993)., Huntley et al., J. Neurosci., 14:3603-3619 (1994), Martin et al., Neuroscience, 53:327-358 (1993), Petralia et al., J. Comp. Neurol., 349:85-110 (1994a), Petralia et al., J. Neurosci, 14:667-696 (1994b), Petralia et al., J. Neurosci., 14:6102-6120 (1994c), Tachibana et al., J. Comp. Neurol., 344:431-454 (1994), Lau et al., J. Biol. Chem., 270:20036-20041 (1995), Roche et al., Neuroscience, 69:383-393 (1995), Craig et al., Proc. Natl. Acad. Sci. USA, 91:2373-12377 (1994), Nusser et al., Neuroscience, 61:421 427 (1994)]. Although both AMPA and NMDA glutamate receptors are clustered at excitatory synapses, neither is present at inhibitory synapses enriched with GABAA receptors [Petralia et al., J. Neurosci., 14:667-696 (1994b), Lau et al., J. Biol. Chem., 270:20036-20041 (1995), Craig et al., Proc. Natl. Acad. Sci. USA, 91:2373-12377 (1994)]. This specific concentration and segregation of excitatory and inhibitory neurotransmitter receptor subunits within a neuron requires innervation of the postsynaptic cell but does not require synaptic activity [Craig et al., Proc. Natl. Acad. Sci. USA, 91:2373-12377 (1994)]. Several recent studies have suggested that NMDA receptors directly or indirectly interact with the neuronal cytoskeleton. For example, actin filament stabilization prevents CA.sup.2+ -dependent inactivation of NMDA channels [Rosenmund et al., Neuron, 10:805-816 (1993)], and NMDA receptor responses are sensitive to cytoskeletal strain [Paoletti et al., Neuron, 13:645-655 (1994)]. In addition, NMDA receptor subunits are enriched in the PSD and are resistant to solubilization in nonionic detergents [Lau et al., J. Biol. Chem., 270:20036-20041 (1995), Brose et al., J. Biol. Chem., 268:22663-22671 (1993)]. Recent experiments have shown that the C-termini of NMDA receptors are involved in the subcellular targeting of the receptors and directly interact with synaptic cytoskeletal proteins [Kornau et al., Science, 269:1737-1740 (1995), Niethammer et al., J. Neurosci, 16:2157-2163 (1996), Ehlers et al., Science, 269:1734-1737 (1995)]. Studies on NMDA receptor expression in fibroblasts have demonstrated that the NR1 subunit is clustered into highly concentrated receptor rich domains near the plasma membrane [Ehlers et al., Science, 269:1734-1737 (1995)]. An alternatively spliced region within the C-terminus of the NR1 subunit, the C1 cassette, is both necessary and sufficient for the localization of NR1 to these receptor-enriched domains, suggesting that the C1 cassette directly interacts with cytoskeletal proteins [Ehlers et al., Science, 269:1734-1737 (1995)]. Interestingly the C1 cassette contains several phosphorylation sites [Tingley et al., Nature, 364:70-73 (1993)] and PKC phosphorylation of this region rapidly disperses the NR1 clusters, perhaps by disrupting the association of NR1 with the cytoskeleton [Ehlers et al., Science, 269:1734-1737 (1995). Kornau et al., Science, 269:1737-1740 (1995)] have recently demonstrated that specific subunits of the NMDA receptor directly interact with the synaptic cytoskeletal associated protein PSD-95 or SAP90 [Cho et al., Neuron, 9:929-942 (1992), Kistner et al., J. Biol. Chem., 268:45804583 (1993)]. PSD95/SAP90 specifically binds to the C-termini of the NR2A, NR2B and NR2D subunits and certain splice variants of the NR1 subunit (NR1d, NR1e) [Kornau et al., Science, 269:1737-1740 (1995), Niethammer et al., J. Neurosci., 16:2157-2163 (1996)]. PSD-95/SAP90 is colocalized with the NR2B subunit in excitatory synapses in neurons and is highly enriched in the postsynaptic density [Kornau et al., Science, 269:1737-1740 (1995), Cho et al., Neuron, 9:929-942 (1992)], where it is well-poised to anchor or target NMDA receptors. PSD-95/SAP90 has an intriguing structure which includes three repeats in the N-terminal region of a newly identified protein motif called a PDZ domain [Cho et al., Neuron, 9:929-942 (1992)], named after three proteins containing this motif, PSD-95, Dlg-A and ZO-1 [Cho et al., Neuron, 9:929-942 (1992), Kennedy, Trends in Biochem. Sci., 20:350 (1995), Gomperts, Cell, 84:659-662 (1996)]. These domains have also been called discs-large-homology regions (DHR) or GLGF repeats (referring to a conserved amino acid sequence in the repeat) [Cho et al., Neuron, 9:929-942 (1992), Kennedy, Trends in Biochem. Sci., 20:350 (1995), Gomperts, Cell, 84:659-662 (1996)]. In addition, PSD95/SAP90 has a arc-homology 3 (SH3) domain and a domain homologous to a yeast guanylate kinase in the C-terminal region [Cho et al., Neuron, 9:929-942 (1992), Kistner et al., J. Biol. Chem., 268:45804583 (1993), Kennedy, Trends in Biochem. Sci., 20:350 (1995), Ponting et al., Trends in Biol. Sci., 20:102-103 (1995), Kim, Cell Biol., 7:641-649 (1995), Gomperts, Cell, 84:659-662 (1996)].
PDZ domains are motifs of approximately 90 amino acids [Cho et al., Neuron, 9:929-942 (1992)] present in a number of homologous proteins including the Drosophila septate junction discs-large protein (Dlg-A) [Woods et al., Cell, 66:451464 (1991)], the mammalian tight junction protein ZO-1 [Itoh et al., J. Cell Biol., 121:491-502 (1993)], the C. elegans epithelial cell junction proteins LIN-2A [Hoskins et al., The C. elegans vulval induction gene lin-2 encodes a member of the MAGUK family of cell junction proteins Development, 122:97-111 (1996)] and Lin-7 [Simske et al., Cell, 85:195-204 (1996)] as well as a variety of other proteins including protein tyrosine phosphatases, nitric oxide synthase, and the mammalian neuromuscular junction protein syntrophin [Ponting et al., Trends in Biol. Sci., 20:102-103 (1995)]. PDZ domains are now thought to mediate a variety of protein--protein interactions [Kornau et al., Science, 269:1737-1740 (1995), Gomperts, Cell, 84:659-662 (1996)].
The interaction of NMDA receptor subunits with PSD-95/SAP90 occurs between the final 7 C-terminal amino acids of the NMDA receptor subunits and the first and second domains of PSD-95 [Kornau et al., Science, 269:1737-1740 (1995), Niethammer et al., J. Neurosci, 16:2157-2163 (1996)]. The common motif present in the C-termini of NMDA receptor subunits which interact with the PDZ domains consists of a threonine or serine followed one amino acid later by a valine (referred to as a T/SXV motif) [Kornau et al., Science, 269:1737-1740 (1995), Niethammer et al., J. Neurosci, 16:2157-2163 (1996)]. Interestingly, such motifs are found in a wide variety of cell surface receptors and ion channels [Kornau et al., Science, 269:1737-1740 (1995), Niethammer et al., J. Neurosci, 16:2157-2163 (1996)]. Indeed, Kim et al. [Kim et al., Nature, 378:85-88 (1995)] have shown that such a motif in the Shaker-type K+-channel subunit Kv1.4 interacts with the PDZ1 and PDZ2 domains of PSD-95/SAP90, with mutations of the conserved threonine completely abolishing the interaction.
It has become increasingly clear that PSD-95/SAP90 is but one of a large family of structurally related proteins. In addition to Dlg-A and ZO-1, which share similar overall domain structures, three additional PSD-951SAP90 family members, SAP97 [Muller et al., J. Neurosci, 15:2354-2366 (1995)], SAP102 [Muller et al., Neuron, 17:255-265 (1996), Lau et al., J. Biol. Chem., 271:21622-21628 (1996)] and PSD-93/Chapsyn [Brenman et al., Cell, 84:757-767 (1996), Kim et al., Neuron., 17:103-113 (1996)] have been identified in the mammalian central nervous system. These homologous proteins may serve functions similar to PSD-95/SAP90 [Ehlers et al., Curr. Opin. in Cell Biol, 8:490495 (1996), Gomperts, Cell, 84:659-662 (1996]. For example SAP102 has recently been shown to interact with NMDA receptor complexes in rat brain [Muller et al., Neuron, 17:255-265 (1996), Lau et al., J. Biol. Chem., 271:21622-21628 (1996)]. A common feature of these PSD-95/SAP90 family members is that they localize to specialized sites of cell-cell contact including septate junctions in Drosophila (Dlg-A), vertebrate tight junctions (ZO-1), and synapses in the mammalian nervous system (PSD-95/SAP90, SAP97, SAP102) [Ehlers et al., Curr. Opin. in Cell Biol., 8:490495 (1996), Kim, Cell Biol., 7:641-649 (1995), Gomperts, Cell, 84:659-662 (1996)]. Members of this family appear to be involved in both localizing cellular proteins and assisting in the establishment of cell polarity. That PSD-95/SAP90 family members might be important for synaptic organization in the mammalian CNS is suggested by the fact that mutations in the homologous Drosophila protein, Dlg-A, alter the structure of glutamatergic synapses [Lahey et al., Neuron., 13(4):823-35 (1994)].